Traditional methodologies for protein detection and quantification include 2-D gel electrophoresis, mass spectrometry and antibody binding. Each methodology has been used to quantify protein levels from relatively large amounts of tissue, yet each suffers from a lack of sensitivity.
Improvement of the ability to monitor proteins, lipids, sugars and metabolite levels and their modifications is needed for cell biology and medicine. A variety of technologies have been employed to improve the sensitivity of detecting these molecules. Recent examples of detection methods include immuno-PCR, RCA and immuno-aRNA.
Immuno-PCR (U.S. Pat. No. 5,665,539) combines the polymerase chain reaction (PCR) technology with conventional detection methods to increase the sensitivity to detect protein. However, a major limitation of immuno-PCR lies in the non-linear amplification ability of PCR reaction, which limits this technique as a quantitative detection method. Thus, this method provides no direct correlation between the amount of signal and the amount of protein present.
A relatively isothermal rolling circle DNA amplification technique (RCA; Schwietzer et al., Proc. Natl. Acad.Sci. USA 97, 10113, 2000)) provides an improvement over immuno-PCR as this technique overcomes some of the quantitation problems associated with immuno-PCR.
U.S. Pat. No. 5,922,553 discloses a method for quantifying levels of a selected protein via a technique referred to as immuno-aRNA. In this method, a first antibody targeted to a selected protein is immobilized to a solid support. The support is then contacted with the selected protein so that the selected protein is immobilized to the first antibody. The solid support is then contacted with a RNA promoter-driven cDNA sequence covalently coupled to a second antibody targeted to the selected protein so that the second antibody binds to the bound selected protein. The amount of selected protein is determined by quantifying levels of the promoter driven cDNA sequence covalently coupled to the bound second antibody via an amplified RNA technique. In a preferred embodiment, a T7 promoter driven cDNA sequence is covalently coupled to the second antibody.
It has now been found that single chain fragments as well as exocyclic peptide based complementarity determining region (CDR) subunits can be used in this immuno-aRNA technique. Further, it has been found that PCR, as well as amplified RNA techniques, can be used to quantify the promoter driven cDNA sequence covalently coupled to the bound single chain fragment or CDR subunit. The use of smaller antibody binding units and fragments coupled with the already existing large single chain or cyclic peptide libraries and the use of robotic assistance renders this method widely useful for both medicinal and research purposes. Furthermore, a single third detector species can be coupled with double-stranded DNA and bound to either the single chain Fv or the CDRs, rendering detection uniform and simple.